In general, existing methodologies for measuring total calcium in biologic fluids involve considerable manipulation of samples and reagents prior to determination of calcium concentration. Gravimetric and titrimetric methods usually require large sample volumes. Colorimetric methods, both manual and automated, commonly involve final readings under highly alkaline conditions; the indicators used for such determinations are unstable at the final pH, thereby requiring reagent dilution with a strong base. Atomic absorption, the methodology recommended by the National Bureau of Standards, is too time consuming to be used in most routine clinical situations.
One commonly-used serum calcium method utilizes cresolphthalein complexone as the indicator. Calcium is dialyzed under acidic conditions into a recipient stream of cresolphthalein complexone solution. A colored complex between calcium and the dye is formed upon the addition of diethylamine which alkalinizes the reaction mixture. The developed color is then measured in a spectrophotometer at 580 nm. In addition to manipulating three separate solutions, the operator must be constantly aware of the condition of the dialyzer membrane. Should the waste solution become acidified through inadvertence or any other reason, deadly hydrogen cyanide gas is generated. Also, practical experience with the system has revealed some problems of drifting baseline and nonreproducibility. Despite these significant disadvantages, such methodology is generally regarded as easier and more predictable than other colorimetric, gravimetric, or titrimetric methods.
Other commercially available procedures also involve handling highly alkaline reagents with the allowable reading (endpoint) being time-dependent. Such procedures nearly always involve an incubation after sample addition and many must be read within a 30 minute time period. In general, commercial procedures for determining total serum calcium do not involve measurements at neutral or acidic conditions.
Ferguson et al., in Analytical Chemistry, Vol. 36, No. 4, 796-799 (April 1964), described the use of chlorophosphonazo III for spectrophotometrically determining calcium and magnesium in non-biologic fluids, specifically, alkaline earth extracts. Unfortunately, the Ferguson et al. teaching that chlorophosphonazo III is a sensitive reagent for selectively determining calcium (and not magnesium) at pH 2.2 is inapplicable to the measurement of calcium in protein-containing body fluids because of the formation of insoluble calcium-protein complexes under such conditions. Subsequent workers, desiring to use chlorophosphonazo III for determining total serum calcium, have therefore developed multiple-step procedures for separating the calcium and protein of the sample before exposing the calcium to the dye. Thus, Howell et al., in Analytical Chemistry, Vol. 38, No. 3, 434-438 (March 1966) disclose first treating the serum sample with tetrabutylammonium oxalate to precipitate all of the calcium from the body fluid. After separation, the precipitate is redissolved in hydrochloric acid and only then is exposed to chlorophosphonazo III.